Heparins, which are mixtures of sulfated mucopolysaccharides of animal origin, are biologically active agents of the glycosaminoglycan family that have particularly useful anticoagulant properties. They are constituted of sulfated linear polysaccharide chains that are very heterogeneous by virtue of their sizes. The average weight of heparins is approximately 15 000 Da (origin: porcine mucus).
Low-molecular-weight heparins (LMWHs) and very low-molecular-weight heparins (VLMWHs) are prepared by cleavage of the long heparin polysaccharide chains into shorter low molecular weight chains. The terms “LMWH” and “VLMWH” are thus intended to mean chains whose molecular weight is respectively between 3000 and 6500 Da and between 1500 and 3000 Da.
Antithrombin III (ATIII) (Chandra et al., 1983, Proc. Natl. Acad. Sci. U.S.A., 80: 1845-1848) is a specific serpin which has a weak inhibitory activity on serine proteases controlling clotting. This action is clearly increased in the presence of heparin which binds and activates ATIII. In particular, the binding to heparin brings about a set of conformational changes in the protein culminating in the adoption of a conformation highly favorable to interaction with the target serine proteases. When ATIII is in the activated conformation, the interaction with the molecule of heparin or derivative which initiated the conformational change is clearly strengthened.
The interaction between heparin and ATIII is due to a specific pentasaccharide sequence. Now, only one-third of the polysaccharide strands have the specific sequences allowing a stable interaction with ATIII. Heparin preparations and derivatives are therefore heterogeneous with regard to the affinity for ATIII. It is important to be able to enrich a population of oligosaccharides in species having affinity for ATIII, such an enrichment being capable of significantly increasing the anticoagulation activity of this population.
A method of ATIII affinity chromatography for separating the heparin-affinity and heparin-non-affinity fractions is described in Hook et al. (1976, FEBS Lett., 66: 90-93). Other methods have been published, but reiterate the essential points of the method of Hook et al. (Hopwood et al., 1976, FEBS Lett., 69: 51-54; Denton et al., 1981, Anal. Biol., 118: 388-391; Pixley & Danishefsky, 1982, Thromb. Res., 26: 129-133). According to this method, ATIII is grafted, in the presence of acetylated heparin, onto CNBr-activated Sepharose B resin. The purpose of using heparin is to prevent any grafting at the level of the heparin-binding site of ATIII.
This technique has two important limitations.
First of all, the heparin used for binding ATIII is an acetylated heparin, so as to avoid the risk of competition with the hexamine NH2 residues. Now, the acetylation of heparin is reflected by a considerable decrease in its affinity and therefore in its protective capacity with respect to the binding site, hence a decrease in the number of ATIII molecules capable of binding the species having affinity.
Secondly, in view of the concentration of ATIII used (approximately 7 mg of protein/ml of hydrated resin), there is a high risk of steric hindrance between the polysaccharide molecules capable of binding to ATIII during a purification of species having affinity for ATIII.